Cells were seeded in 60 mm Petri dishes (Thermo Fisher Scientific, Roskilde, Denmark) at a density of 720 to 7200 cells per dish and incubated for 24 h at 37 °C before the addition of the KIs CC115 (1 μmol/L), AZD7648 (5 μmol/L), or Sapanisertib (0.5 μmol/L). We delivered 2 Gy IR 3 h later by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After 24 h of incubation, the medium containing the drug was replaced with fresh medium. Cultures were incubated for 1–3 weeks until colonies were formed. Colonies were stained with methylene blue (#66725, Sigma Aldrich, Munich, Germany) for 30 min at room temperature. Clusters containing 50 or more cells were considered to be a colony. Image analysis software (Biogas Kobi 1.2) was used to count the colonies (Biomas, Erlangen, Germany). The determination of plating efficiency (PE) involved calculating the ratio of the colonies formed to the initially seeded cells. The survival fraction (SF) was then computed by dividing the number of colonies formed by the PE and multiplying the result by the number of cells initially seeded [59 (link)].
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